Colorimetric method for determining iron in blood



United States Patent 3,506,404 COLORIMETRIC METHOD FOR DETERMINING IRONIN BLOOD George Evans, Hopatcong, and Ronald Searcy, Upper Montclair,NJ., assignors to Hotfmann-La Roche Inc., Nutley, N.J., a corporation ofNew Jersey No Drawing. Filed Dec. 19, 1967, Ser. No. 691,703

Int. Cl. G01n 31/22 U.S. Cl. 23-230 14 Claims ABSTRACT OF THE DISCLOSUREBACKGROUND OF THE INVENTION Techniques have been developed forquantitatively determining the iron content of whole blood and serum bymeans of reacting the iron in either its ferric or ferrous state withvarious color forming reagents such as ammonium thiocyanate,bathophenanthroline, dipyridyl, etc. However, this process has sufferedfrom several disadvantages due to the fact that known color formingreagents are too sensitive to extraneous sources so that they pick uptrace contaminants. In many cases these color forming reagents do notproduce a sufficient color difierentiation between the blank and thesample prepared from whole blood or serum to be tested. This makes thequantitative determination of the iron content in this sample ditficultto carry out by standard colorimetric instruments such as a BeckmanSpectrophotometer.

The colorimetric technique for quantitating iron has been important indetermining the hemoglobin content of whole blood since by utilizing astandard conversion factor the hemoglobin present in whole blood can becalculated from the iron content of the blood sample. However, theliberation of the iron from the hemoglobin to place it in a watersoluble state and in a state where it can be quantitatedcolorimetrically has proven too complicated and time-consuming forroutine diagnostic use. This procedure of liberation and solubilizationof the iron from the hemoglobin involves releasing iron in a watersoluble state by first adding concentrated sulfuric acid and potassiumpersulfate at room temperature, and thereafter removing the protein byprecipitation by the addition of sodium tungstate.

The procedure for liberating iron from serum in a water soluble state,where the quantity of iron can be determined by use of a color formingreagent, has also proven to be too tedious and complicated for routinediagnostic use since the iron was liberated from the serum by ashing.

SUMMARY OF THE INVENTION In accordance with this invention we havediscovered that when a compound selected from the group consisting ofcompounds of the formula:

wherein A is selected from the group consisting of and (;=N r'a 8 B isselected from and -CH R is selected from the group consisting ofhalogen, hydrogen, trifluoromethyl, nitro and amino; R is selected fromthe group consisting of H -R7-( ]-R3 hydrogen, lower alkyl and R0--CI1H2nN n is an integer from 2 to 7; R is selected from the groupconsisting of hydrogen, hydroxy, lower alkyl, lower alkoxy and loweralkanoyloxy; R is 2-pyridyl; R is selected from the group consisting oflower alkyl and hydrogen; R is selected from the group consisting oflower alkyl, hydrogen,

and R and R where taken together with their attached nitrogen atom forma radical selected from the group consisting of piperazinyl, lower alkylsubstituted piperazinyl, pyrrolidinyl, lower alkyl substitutedpyrrolidinyl, piperidinyl and lower alkyl substituted piperidinyl; R7 islower alkyl; and R is selected from the group consisting of lower alkyland hydrogen and water soluble salts thereof are added to an aqueoussolution containing ferrous ions prepared from whole blood or serum, apurple colored solution is obtained which can be quantiated as to itsiron conent by standard colorimetric means.

The color differentiation with different concentrations of ferrous ionsproduced by the compound of Formula I above, is such that theconcentration of iron therein can be easily determined by standardcolorimetric instruments. Furthermore, the compound of Formula I is notsensitive to extraneous sources so as to include interferences fromtrace contaminants of iron. Therefore, the method of this inventionprovides a simple colorimetric means of quantitatively determining theiron content of blood serum and whole blood.

By means of utilizing the compound of Formula I above, as a colorforming reagent, the iron content of hemoglobin can be quantitativelydetermined by colorimetric means without the necessity of first treatingthe whole blood sample with a mixture of sulfuric acid and potassiumpersulfate and then removing the protein by the addition of sodiumtungstate. In accordance with this invention, the hemoglobin in a wholeblood sample can be determined by first extracting the iron into anaqueous solution in the ferric state from the hemoglobin by addition ofsodium hypochlorite, thereafter reducing the ferric ions to ferrousions, reacting the ferrous ions with the compound of Formula I above toproduce a brilliant deep color and colorimetrically quantitating theiron present in the solution. The quantity of hemoglobin present thereincan be calculated utilizing a standard conversion factor based upon thequantity of iron present in the hemoglobin molecule. As can be seen, theabove procedure provides a simple and quick method for quantitativelydetermining the hemoglobin content of whole blood, which is ideallysuited for routine diagnostic use.

In the case of blood serum, the iron content can be deter-mined by useof the compound of Formula I without the necessity of ashing in order toextract the iron from serum into an aqueous solution. In accordance withthis invention, the iron is first liberated from the serum in an aqueoussolution in the ferric state 'by extraction with an acid deproteinizingagent. The ferric ion 15 then reduced to the ferrous state. The ferrousion in the aqueous solution is then reacted with the compound of FormulaI above the produce a brilliant deep purple color. The iron content inthe resulting purple solution can be quantitatively determined bystandard colorimetric means.

DETAILED DESCRIPTION OF THE INVENTION The term lower alkyl as usedthroughout this specification includes both straight and branched chainalkyl groups having from 1 to 7 carbon atoms such as methyl, ethyl,propyl, isopropyl and the like. The term lower alkanoyloxy refers toboth straight chain and branched chain aliphatic carboxylic acidmoieties, such as acetoxy, propionyloxy, butyrolxy and the like. Theterm halogen includes bromine, chlorine, fluorine and iodine. Alsoincluded within the purview of the present invention are the watersoluble acid addition salts of the compounds of Formula I above. Anyconventional water soluble acid addition salts of the compounds ofFormula I above, may be utilized in the process of this invention toquantitatively determine the iron content of aqueous solutions. Amongthe acid addition salts which can be utilized 1n accordance with thisinvention, includes salts of compounds of the Formula I with organic orinorganic ac ds such as hydrochloric acid, hydrobromic acid, nitric acd, sulfuric acid, acetic acid, formic acid, succinic acld, maleic acid,p-toluene sulfonic acid and the like.

Examples of benzodiazepine compounds of Formula I above which areparticularly suitable as the color forming reagent in the process ofthis invention include the following: 7-bromo-1,3-dihydro-1 [4-(4-methyll-piperazinyl) butyl]--(2-pyridyl)-2H-1,4-benzodiazepin-2-one;7-amino-1,3-dihydro-5-(2-pyridyl) 2H-1,4-benzodiazepin-Z-one; l-methyl-1- 3- (7-bromo-5- 2-pyridyl) -l ,3-dihydro-2-oxo-2H-1,4-benzodiazepin-1-yl)propyl]urea whose preparation is disclosedin US. patent application Ser. No. 677,092, filed Oct. 23, 1967 in thename of Barley et al.;

7-bromo-1,3-dihydro-5-(2-pyridyl)-2H-1,4-

benzodiazepine; 7-amino-l,3-dihydro-l-methyl-S-(Z-pyridyl)-1H-1,4-benzodiazepine; 7-bromo-1,3-dihydro-(3 -dimethylaminopropyl) -5-(2-pyridyl)-2H-1,4-benzodiazepin-2-one;7-bromo-l,3-dihydro-5-(2-pyridyl)-2H-1,4-

benzodiazepin-Z-one 4-oxide; 7-bromo-1,3-dihydro-S-(Z-pyridyl)-2H-1,4-

benzodiazepi-n-Z-one; 7 bromo-1,3-dihydro-l-(B-hydroxypropyl)-5-(2-pyridyl(2H-l,4-benzodiazepin-2-one; and7-bromo-5-(2-pyridyl)-1,3-dihydro-1-[3-(N-cyanomethylamino)propyl]-2H-1,4-benzodiazepin-2-onewhose preparation is disclosed in US. patent application Ser. No.677,092, filed Oct. 23, 1967 in the name of Barley et al.

By utilizing the color forming reagent of Formula I above, one canquantitate the iron content of serum without the necessity of utilizingashing to liberate or extract the iron from the serum. In accordancewith this invention, iron can be liberated from serum by means oftreating the serum with an acidic deproteinizing agent. The acidicdeproteinizing agent extracts the iron from the'serum in the ferricstate. In extracting iron from the blood serum, any conventional acidicdeproteinizing agent such as trichloroacetic acid, nitric acid orperchlo'ric acid can be utilized.

The aqueous solution which contains the free iron in the ferric state isthen treated with a chemical reducing agent to convert the iron to thedesired ferrous state. In -general any known chemical reducing agent canbe used in this step of the procedure. These include, for example,ascorbic acid, hydrazine sulfate, thioglycolic acid, sodiumthioglycolate, sodium hydrosulfite, sodium metabisulfite, sodiumbisulfite, sodium sulfite, hydroxylamine hydrochloride, hydroxylaminesulfate, hydroquinone, stannous chloride, etc. In the preferredembodiment of the invention, however, the chemical reduction of freeiron from the ferric state to the ferrous state is carried out usingthioglycolic acid or ascorbic acid as the reducing agent. The use ofthioglycolic acid is advantageous since it permits the reduction of thefree iron from the ferric state to the ferrous state to be accomplishedat room temperature. The quantity of thioglycolic acid which is used inthis step of the process is not particularly critical. A sufiicientamount of the reducing agent should be used however to completelyconvert the free iron to the ferrous state. An excess of thioglycolicacid can be used, if desired, to insure complete conversion.

In a whole blood sample, the hemoglobin is determined by firstliberating the iron from the fresh whole human blood in an aqueoussolution in the ferric state. In general, the iron can be liberated fromthe sample by any convenient and conventional method. However, inaccordance with this invention, the liberation of the iron can beaccomplished by treating the blood at room temperature with ahypochlorite salt, preferably sodium hypochlorite. The use of ahypochlorite salt serves to liberate the iron in the form of ferric ionswhich when reduced to ferrous ions and then reacted with the compound ofFormula I, will produce a color that can be quantitated so that theamount of iron in the blood can be determined. Any of the conventionalmethods of reducing the ferric ion liberated from the blood sample tothe ferrous state such as hereinbefore described, can be utilized.

The quantity of hypochlorite salt which is used in the preferredembodiment of this invention is variable. However, a sufficient quantityof hypochlorite salt should be used to liberate all of the iron which ispresent in the blood. An excess of hypochlorite salt can be used, ifdesired, to insure that all of the iron present in this sample isliberated. The precise manner in which the hypochlorite salt isincorporated into the sample is not particularly critical. Generally, itwill be introduced into the sample in the form of a dilute aqueoussolution. In the preferred practice of this invention, the salt is addedto the sample in the form of an aqueous solution containing from about 5percent to about 25 percent by weight of a hypochlorite salt such assodium hypochlorite.

When the iron has been liberated from the blood sample to be quantitatedin the water soluble ferrous state, the compound of Formula I above isadded to an aqueous solution containing the extracted ferrous ions. Uponmixing, a complexing takes place between the ferrous ion and thecompound of Formula 1 above. The formation of the complex is evidencedby the development of a brilliant purple color. The precise manner inwhich the compound of Formula I above is added to the ferrous ionsolution does not limit the scope or practice of this invention.Generally, however, the compound of Formula I above is added to thesystem in the form of a relatively dilute aqueous solution. In thepreferred practice of the invention, there is used an aqueous solutioncontaining from about 0.25 percent to about 0.75 percent by weight ofthe compound of Formula I above.

The quantity of the compound of Formula I, which is added to the aqueoussolution is variable. In all instances, however, a suflicient quantityof the compound of Formula I above, should be provided to react with allof the ferrous ions present in the aqueous solution. In order to insure,however, that all of the ferrous ions present in the mixture have beencomplexed, it is preferred to add a quantity of the color formingcompound of Formula I above, which is in excess of that actuallyrequired to complex all of the ferrous ions which are available forcomplexing.

The desired purple coloration of the mixture will be noted immediatelyupon the addition of the compound of Formula I above, to the system. Thecolor deepens as the reaction proceeds to completion. After standing forawhile at room temperature, color changes are no longer discernible tothe naked eye. Accordingly, in order to in sure uniform coloring, theaqueous solution should be allowed to stand until its color appears tohave become constant. In general, it has been found that fulldevelopment of the purple color will occur over a period of from about 5to minutes.

The quantitation of the iron in the colored sample can be carried out byany conventional colorimetric method utilizing standardspectrophotometers such as a Beckman Spectrophotometer, Coleman Juniorspectrophotometer, etc.

The complex which is produced in the practice of the invention has itsown spectral characteristics. Color was developed using a samplecontaining 6.0 g. of iron. The optical density of the mixture was thenmeasured at wave lengths ranging from 450-700 m in a BeckmanSpectrophotometer, Model DBG, using a 1 cm. cuvette. With increasingwave length above 450 m there was a progressive rise in absorbance whichreached a maximum at about 580 m The absorbance diminished at higherwave lengths.

In order to determine the speed and stability of the colorimeterresponse, a sample containing 6.0 g. of iron was prepared. This samplewas transferred to a 1 cm. cuvette immediately after the benzodiazepinesalt color reagent had been added thereto. For a period of about onehour, optical density measurements were made continuously at 5 80 m Itwas found that the absorbance increased rapidly reaching a maximum valuein about seven minutes and, further, that the measurement changed littlethereafter.

The quantitative determination of iron in the sample is carried out asfollows: the optical density of the purple colored sample is measuredagainst a reagent blank at 580 m using a standard spectrophotometer,e.g. a Coleman Junior Spectrophotometer, employing a cuvette with 19 mm.light path. The quantity of iron in the specimen is determined in theconventional manner from the absorbance of the specimen with referenceto a standard. The iron content in a sample such as serum is calculatedaccording to the following formula:

Iron content of sample (mcg./ 100 ml.)

absorbance of Sample absorbance of Standard iron concentration ofstandard (mcg./ 100 ml.)

In determining the hemoglobin content of a whole blood sample, basedupon the iron content thereof, the following formulation can beconveniently utilized Hemoglobin in sample (grams/ 100 ml.)

=hemoglobin in standard (grams/100 ml.)

absorbance sample absorbance standard The hemoglobin in the standard iscalculated by the following formula:

Hemoglobin in standard (grams/ ml.)

Iron in standard (milligrams/ 100 ml.) 3.47

The constant 3.47 is used for the conversion since it is now acceptedthat hemoglobin contains 0.347 percent of iron.

As indicated heretofore, the present invention provides an extremelyimportant diagnostic and analytical tool. The described method can heused to determine the iron in various materials such as body fluidsrapidly and accurately. More particularly, the method can be used toquantitate the iron in blood serum or to determine the hemoglobin infresh whole human blood based on its iron con tent. In addition to beinga rapid and accurate method for making the determination, the resultsobtained by the test method are characterized by a high degree ofreproduci'bility.

For a fuller understanding of the nature and objects of this invention,reference may be had to the following examples which are given merely asfurther illustrations of the invention and are not to be construed in alimiting sense.

Example 1 This example demonstrates the applicability of the test methodto a blood hemoglobin determination.

In the method, a 20 l. volume of whole blood was added to 2.0 ml. of anaqueous solution containing 17 percent by weight of sodium hypochlorite.To the mixture, thus obtained, was added 6.5 ml. of 2.7 percentthioglycolic acid prepared in 1 M sodium acetate. Subsequently, 1.5 ml.of an aqueous solution containing 0.33 percent of 7-bromo 1,3 dihydro 1(3-dimethylaminopropyl)- 5-(2-pyridyl) 2H 1,4 benzodiazepin-Z-onedihydro chloride was added to the mixture. Almost immediately, a purplecoloration was noted. The reaction mixture was allowed to stand for aperiod of about ten minutes to allow the color to fully develop. Theoptical density of the mixture was measured against a reagent blank at580 m in a Coleman Junior Spectrophotometer using a cuvette with 19 mm.light path.

Using the data thus obtained, the hemoglobin content of the blood samplewas calculated using the following formula:

Hemoglobin in sample (grams/100 ml.)

=hemoglobin in standard (grams/100 ml.)

- absorbance sample absorbance standard The hemoglobin in the standardwas calculated using the following formula:

Hemoglobin in standard (grams/100 ml.)

iron in standard (milligrams/100.) 3.47

Test procedures the benzodiazepine, i.e., 7-bromo-1,3- dihydro 1(3dimethylaminopropyl) 5 (2-pyridyl)- 2H-l,4-benzodiazepin-2-onedihydrochloride, described in the preceding paragraph were performed onblood samples obtained from healthy individuals and hospitalizedpatients. For comparative purposes, the hemoglobin content of the sameblood samples was also determined by the cyanmethemoglobin method. Inthe latter procedure, a 20 pl. volume of whole blood was added to 5.0ml. of an aqueous solution containing 1.0 gram per liter of sodiumbicarbonate, 50.0 mg. per liter of potassium cyanide and 200 mg. perliter of potassium ferricyanide. After allowing a period of ten minutesfor complete color formation, the optical density of each sample wasmeasured against a reagent blank at 540 mp in a Coleman JuniorSpectrophotometer using a cuvette with a 19 mm. light path. The netabsorbance was converted into hemoglobin concentrations by means of astandard curve constructed with dilutions of cyanmethemoglobin of knownconcentration.

The results obtained utilizing the aforesaid two techniques are setforth in the following table:

TABLE-DETERMINATION. F HEMOGLOBIN CONTENT Example 2 To a stirredsolution of 19 g. (0.06 mole) of 7-bromo- 1,3- dihydro--(2-pyridyl) 2H1,4 benzodiazepin-Z- one in 200 ml. of dry N,N-dimethylformamide wasadded portionwise 3 g. (0.063 mole) of 50% sodium hydride. The resultantsolution was then cooled with an ice-water bath while 39 g. (0.18 mole)of 1,4-dibromobutanewas added dropwise during 25 min. After stirringovernight at room temperature the solution was poured into 600 ml. ofwater and extracted with methylene chloride. The organic layer wasseparated, washed with brine, dried over sodium sulfate andconcentrated. The resultant oil was dissolved in ether and then filteredfrom a small amount of precipitated solid (discarded). This filtrate wasthen put on silica gel and the silca gel washed with ether. The first150-200 ml. of eluant was separated while the subsequent fractions (2 1.total) were concentrated to give 8 g. of a tan colored solid. The firstether fraction upon concentration gave an oil which was dissolved in asmall amount of ether and seeded with a crystal from the 8 g. sample togive 7-bromo-1-(4-bromobutyl)-5-(2-pyridyl)-1,3-dihydro-2H-1,4-benzodiazepin-2-one.

EXAMPLE 3 A mixture of 9 g. (0.02 mole) of7-bromo-1-(4-bromobutyl)-5-(2-pyridyl) 1,3dihydro-ZH-1,4-benzodiazepin-2-one, 3 g. (0.03 mole) ofN-methylpiperazine, 3 g. (0.02 mole) of sodium iodide and 100 ml. ofmethylethyl ketone was stirred and refluxed for 22.5 hrs. Solvents wereremoved at reduced pressure and the residue partitioned betweenmethylene chloride and water. The methylene chloride layer was washedwith brine, dried over sodium sulfate and concentrated. Upon theaddition of ether to the resultant oil a solid separated. The suspensionwas filtered and the solid discarded. The ether filtrate wasconcentrated and the residual oil was converted to the dihydrochlorideby the addition of the the theoretical amount of methanolic hydrogenchloride followed by ether. The resultant highly hygroscopic solid wasrecrystallized from ethanol-ether to give7-bromo-l,3-dihydro[4-(4-methyl-l-piperazinyl)butyl] 5 (2-pyridy1)-2H-1,4-benzodiazepin-2-one dihydrochloride as white needles.

7-bromo-l,3-dihydro[4-(4- methyl 1 piperazinyl) bntyl] -5-(2 pyridyl)-2H-1,4 benzodiazepin-2-one dihydrochloride was utilized in the samemanner as 7-bromo- 1,3 dihydro-l-(3-dimethylaminopropyl)-5-(2.-pyridyl)-2H-l,4-benzodiazepin-2-one dihydrochloride to quantitate the iron inserum and the hemoglobin content of whole blood. The procedure utilizedwas the same as given in Example 1.

EXAMPLE 4 This example demonstrates the applicability of the test methodto a serum iron determination.

The iron content in five different samples of serum was determined byutilizing the following procedure: 2 milliliters of clear unhemolyzedserum and 1.0 milliliters of 2 N hydrochloric acid was allowed to standfor 20 minutes. After standing, 3 milliliters of 17 percent by weighttrichloro acetic acid aqueous solution was added, under constantstirring, to the serum. After adding the trichloro acetic acid, theserum was centrifuged at 2,500 r.p.m. for 15 minutes. 4 millilitersof'the clear supernatant was treated with 2.0 milliliters of a 25percent by weight ammonium acetate and 75 percent by weight watersolution which contained 0.1 percent by weight of 7-bromo 1,3dihydro-1-(3-dimethyla minopropyl)-S-Z-pyridyl-ZH-l,A-benzodiazepin-Z-one dihydrochloride. The opticaldensity of the mixture was measured against a reagent blank at 5 mg in aColeman Junior Spectrophotometer using a cuvette with a 19' mm. lightpath. The iron content of the serum sample was calculated in the mannerhereinbefore mentioned.

The iron content of these 5 samples was also determined by the methodgiven by D. S. Fisher et a1. utilizing tripyridyltriazine as describedin Clin. Chem, 10: 21, (1964).

The results obtained utilizing the aforesaid two techniques are setforth in the following table:

DETERMINATION OF SERUM IRON CONTENT (pg) Benzodiazepine test Sample No.(pg/ m1.) Fisher et a1.

We claim:

1. A process for quantitating the iron content of whole bloodcomprising:

(a) extracting the iron in said blood into an aqueous solution in theferric state;

(b) treating the ferric ions in said aqueous solution with a reducingagent to reduce the ferric ions'to ferrous ions;

(c) reacting the ferrous, ions with a compound selected from the groupconsisting of compounds of the formula and --CH R is selected from thegroup consisting of halogen, hydrogen, trifl-uoromethyl, nitro andamino; R is selected from the group consisting of 10 hydrogen, loweralkyl and n is an integer from 2 to 7; R is selected from the groupconsisting of hydrogen, hydroxy, lower alkyl, H2 lower alkoxy and loweralkanoyloxy; R is 2-pyridyl; R is selected from the group consisting oflower Rs 5 alkyl and hydrogen; R is selected from the group n is aninteger from 2 to 7; R is selected from the consisting of lower y y ggroup consisting of hydrogen, hydroxy, lower alkyl,

lower alkoxy and lower alkanoyloxy; R is Z-pyridyl; R is selected fromthe group consisting of lower alkyl and hydrogen; R is selected from thegroup 10 f and e and e Where taken tqgether consisting f lower alkylhydrogen, with their attached nitrogen atom form a radical selected fromthe group consisting of piperazlnyl, lower alkyl substitutedpiperazinyl, pyrrolidinyl, lower alkyl substituted pyrrolidinyl,piperidinyl and lower alkyl and CEN; and R and R where taken togetherSubstituted p p y 1 is lowfil' y and s is with their attached nitrogenatom form a radical Selected from the g p Consisting of lower alkyl andselected from the group consisting of piperazinyl, hydrogen low alkyl btit t d i era i l, rrolidinyl, and water soluble acid addition saltsthereof to color said lower alkyl substituted pyrrolidinyl, piperidinyland solution; and lower alkyl substituted piperidinyl; R is lower alkyl;(d) colorimetrically quantitating the iron content of and R is selectedfrom the group consisting of said solution by means of said color.

lower alkyl and hydrogen 6. The process of claim 5 wherein said compoundis 7- and water soluble acid addition salts thereof to produce br y -1-(yl minopropyl)-5-(2 pyrcolored solutions; andidyl)-2H-l,4-benzodiazepin2-one.

(d) colorimetrically quantitating the iron present in The process ofclaim 5 wherein said compound is 7- the solution by means of said color.bromo-1,3-d y 0- y -p p yn y l' 2. The process of claim 1 wherein theiron is ex- S-( -PY Y P -2- tracted from the whole blood by the additionof sodium 8. The process of claim 5 wherein said iron is extractedhypochlorite. with an acid deproteinizing agent.

3. The process of claim 1 wherein said compound is 9. The process ofclaim 8 wherein said deproteinizing 7-bromo 1,3dihydro-l-(3-dimethylaminopropyl-5-(2- agent is trichloroacetic acid.pyridyl)-2H-l,4-benzodiazepin-2-one; 10. A process for quantitating thehemoglobin content 4. The process of claim 1 wherein said compound is ofwhole fresh blood comprising: l7-bromo-1,3-dihydro-l-[4-(4-methyl-l-piperazinyl)butyl]- (a) extractingthe iron in said blood into an aqueous5-(2-pyridyl)-2H-l,4-benzodiazepin-2-one. solution in the ferriq state;

5. A process of quantitating the iron content in serum (b) treating theferric ions in said aqueous solution with comprising: a reducing agentto reduce the ferric ions to ferrous (a) extracting the iron from saidserum into an aqueous ions;

olution in th f ri tate; (c) reacting the ferrous ions with a compoundselected (b) treating said solution with a reducing agent to 40 from thegroup consisting of compounds of the forreduce the ferric ions to theferrous state; a

(-c) reacting the ferrous ions in said solution with a compound selectedfrom the group consisting of compounds of the formula H R1 o NB /H A/ R3B1 wherein A is selected from the group consisting of \A/ R: C=N whereinA is selected from the group consisting of and 1'1, 8 and B. l f

1s se ected rom l J 0 R4 0 H B is selected from O and CH;; R 18 selectedfrom the group consisting of halogen, hydrogen, trifluoromethyl, nitroand amino; R is selected from the group consistand CH R is selected fromthe group consisting of ing of halogen, hydrogen, trifluoromethyl, nitroand H amino; R is selected from the group consisting of V f n hydrogen,lower alkyl and H a hydrogen, lower alkyl and /Ra R5 ---0 H N n m n isan integer from 2 to 7; R 1s selected from the R1 group consisting ofhydrogen, hydroxy, lower alkyl,

ii lower alkoxy and lower alkanoyloxy; R is 2-pyridy1; R is selectedfrom the group consisting of lower alkyl and hydrogen; R is selectedfrom the group consisting of lower alkyl, hydrogen,

- -NHz and CEN; and R and R where taken together with their attachednitrogen atom form a radical selected from the group consisting ofpiperazinyl, lower alkyl substituted piperazinyl, pyrrolidinyl, loweralkyl substituted pyrrolidinyl, piperidinyl and lower alkyl substitutedpiperidinyl; R is lower alkyl; and R is selected from the groupconsisting of lower alkyl and hydrogen and water soluble acid additionsalts thereof to produce colored solutions;

(d) colorimetrically quantitating the iron present in the solution bymeans of said color; and

(e) calculating the hemoglobin content based upon the quantitativedetermination of the iron present in said solution.

11. The process of claim 10 wherein said compound is 7-bromo 1,3dihydro-1-(3-dimethylaminopropyl)-5-(2-pyridyl)-2H-1,4-benzodiazepin-2-one.

12. The process of claim 10 wherein said compound is7-bromo-l,3-dihydro-1-[4-(4-methy1-1-piperazinyl)butyl]-5-(2-pyridyl)-2I-I-1,4-benzodiazepine.

13. The process of claim 10 wherein said compound is7-bromo-1,3-dihydro-5-(2-pyridyl)-2H-1,4-benzodiazepin- 2-one.

14. The process of claim 10 wherein said iron is extracted by theaddition of an aqueous hypochlorite salt.

References Cited UNITED STATES PATENTS 3,100,770 8/1963 Fryer et a1260239.3 3,182,066 5/1965 Fryer et a1 260-239.3 3,236,838 2/1966 Archeret a1 260-239.3 3,299,053 1/ 1967 Archer et al 260239.3

OTHER REFERENCES Henry, R. J. et al., Chemical Abstracts, vol. 53, p.4403 1959 Welcher, I. 1., edition, Standard Methods of ChemicalAnalysis, vol. 1, pp. 528-; vol. 2, pp. 1098-1103 (1963).

MORRIS O. WOLK, Primary Examiner E. A. KATZ, Assistant Examiner US. Cl.X.R.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No.506,401; Dated April 14, 1970 Inv Evans and Searoy It is certified thaterror appears in the above-identified patent and that said LettersPatent are hereby corrected as shown below:

Column 8, lines 72-75 R3 should be R-7- ir- R8 OH OH Column 9 lines 677O R; c -R should be RT 0-11 OH OH cum MD 32212.20 JAN 5 1971 ISEAL)Attest:

Flmhm mm x. mm, m.

Officer fifimissioner of Pat out;

